JPH07242599A - Interleukin-1-producing inhibitor - Google Patents

Abstract

PURPOSE:To obtain an interleukin-1-producing inhibitor containing a 2- alkynylmalonic acid derivative as an active ingredient and useful for treating chronic rheumatism or inflammatory diseases. CONSTITUTION:This interleukin-1-producing inhibitor contains a compound of the formula (R is H, an aryl, an aralkyl, a cycloalkyl, a cycloalkenyl, a lower alkyl, etc.; R<1> is H or a carboxyl-protecting group; R<2> is OH, a lower alkoxy, an amino, etc.) or its salt as an active ingredient. The compound of the formula has an activity close to prednisolone which is a conventional excellent steroidal compound and has inhibition activity equivalent to or higher than that of tenidap which is a nonsteroidal compound. The inhibitor is useful for arthrosis deformans, gout, atherosclerosis, atopic dermatitis, lung diseases accompanied by granuloma, various encephalitis, diabetes mellitus and osteoporosis and is effective also as immunosuppressive agent.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an interleukin 1 containing a 2-alkynylmalonic acid derivative or a salt thereof.
It relates to a production inhibitor.

[0002]

BACKGROUND OF THE INVENTION Various cytokines and chemical mediators are involved in the regulation of immunity in the body of animals. Among these cytokines, interleukin 1 (hereinafter referred to as IL-1) produced from monocyte cell types and the like
Acts on various cells and plays an important role in many biological reactions such as immunity, inflammation, hematopoiesis, endocrine, and cranial nerves (Therapeutics vol24 NO1, 1990, etc.). Currently, these IL-1s are known to exist in the pI5 type and the pI7 type due to the difference in isoelectric point. Autoimmune diseases and inflammatory diseases are closely related to this abnormal production of IL-1. For example, it has been reported that abnormal production of IL-1 in rheumatoid arthritis is associated with fever, cartilage destruction, bone destruction, and synovial cell proliferation. In fact, there is a report that IL-1 is excessively present in the synovial fluid of rheumatoid patients, and peripheral blood monocytes of active rheumatic patients have enhanced IL-1 producing ability.
Other autoimmune diseases (systemic lupus erythematosus,
It is said to be related to Chet's disease, scleroderma, atopic dermatitis, etc.), osteoporosis, diabetes, etc. On the other hand, as a therapeutic agent for these rheumatoid arthritis, prednisolone is known to have the most excellent therapeutic effect than before, but it is a steroidal compound, and it has a full moon-like face, induced infection, and secondary adrenal cortex function. There are problems of side effects such as insufficiency, peptic ulcer, increased appetite, and hirsutism, and improvement is desired. Non-steroidal anti-inflammatory agents are also used, but they are nothing but so-called symptomatic treatments, and the occurrence of side effects due to the mechanism of action is unavoidable.

Therefore, attempts have recently been actively made to use a compound having an IL-1 production inhibitory effect as a therapeutic agent for rheumatoid arthritis or autoimmune diseases (Japanese Patent Laid-Open No. 256656/1990, Europe). Publication EP5969
8 or JP-A-3-133929). Further, although some of the compounds according to the present invention are known, these 2-alkynylmalonic acid derivatives are IL
It is not known to have a -1 production inhibitory action.

[0004]

The object of the present invention is to provide an excellent inhibitor of IL-1 production without side effects, and the present inventors have conducted extensive studies on an inhibitor of IL-1 production. As a result, the IL of the 2-alkynylmalonic acid derivative was excellent.
The present invention was completed by discovering that it has a -1 production inhibitory action.

[0005]

[Means for Solving the Problems] That is, the present invention provides the following IL-1 production inhibitor, IL-1β production inhibitor,
The present invention relates to a therapeutic agent for rheumatoid arthritis and a therapeutic agent for autoimmune diseases. (1) The following general formula [I]

[0006]

[Chemical 1]

(Wherein R represents a hydrogen atom, an optionally substituted aryl group or aralkyl group, a cycloalkyl group, a cycloalkenyl group, an optionally substituted lower alkyl group or a tri-lower alkylsilyl group, R ¹ represents a hydrogen atom or a carboxyl protecting group, and R ² represents a hydroxyl group, a lower alkoxy group, an amino group which may be protected, or an alkylamino group which may be protected) 2-alkynylmalonic acid An interleukin-1 production inhibitor comprising a derivative or a salt thereof. (2) An interleukin-1β production inhibitor comprising the 2-alkynylmalonic acid derivative or the salt thereof according to (1) above. (3) A therapeutic agent for rheumatoid arthritis, which comprises the 2-alkynylmalonic acid derivative or the salt thereof according to (1) above. (4) A therapeutic agent for an autoimmune disease, which comprises the 2-alkynylmalonic acid derivative or the salt thereof described in (1) above.

The meanings of the terms relating to the substituents in this specification are as follows. "Aryl group" means
It means an aromatic hydrocarbon group such as a phenyl group, a 1-naphthyl group, a 2-naphthyl group and a biphenyl group, preferably a phenyl group, a 1-naphthyl group and a 2-naphthyl group, particularly preferably a phenyl group. . What is an "aralkyl group"?
It means a phenyl lower alkyl group such as a benzyl group and a phenethyl group, and a benzyl group is particularly preferable. These "aryl group" and "aralkyl group" are lower alkyl groups such as methyl group, ethyl group, propyl group and isopropyl group;
Hydroxyl group: It may be substituted with one or more substituents such as a lower alkoxy group such as a methoxy group, an ethoxy group and a propoxy group. The “cycloalkyl group” is a saturated aliphatic hydrocarbon ring having 3 to 7 carbon atoms, and specifically means a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group or a cycloheptyl group. Preferred are cyclopropyl group, cyclobutyl group, cyclopentyl group and cyclohexyl group, and particularly preferred are cyclopentyl group and cyclohexyl group. The "cycloalkenyl group" means, for example, a cyclopentenyl group, a cyclohexenyl group or the like in which at least one pair of adjacent carbon atoms in the saturated aliphatic hydrocarbon ring of the cycloalkyl group as described above are linked by a double bond. means. "Lower alkyl group" means
It means a linear or branched alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and a tert-butyl group. These "alkyl groups" include a hydroxyl group; a protected hydroxyl group; a mercapto group; a carboxy group; a substituent such as a lower alkoxycarbonyl group or a 5-dimethylamino-1-naphthalenesulfonyloxy group, preferably a hydroxyl group, a protected hydroxyl group. May be replaced with. The "hydroxyl-protecting group" may be any group that can be eliminated by hydrolysis or hydrogenolysis, and is, for example, a tetrahydropyranyl group. The "tri-lower alkylsilyl group" is, for example, a trimethylsilyl group or a triethylsilyl group. The "carboxyl-protecting group" may be any group as long as it substantially protects the carboxyl group, but is particularly preferably a lower alkyl group such as methyl group, ethyl group or aralkyl group. The "amino protecting group" in the optionally protected amino group or optionally protected alkylamino group is an amino protecting group used in the field of ordinary amino acid chemistry, and protects the amino group or imino group from various reactions. Any protecting group can be used as long as it does. For example, substituted or unsubstituted lower alkanoyl such as formyl group, acetyl group, propionyl group, trifluoroacetyl group; phthaloyl group; lower alkoxycarbonyl group such as tert-butoxycarbonyl group (Boc); benzyloxycarbonyl group (Z) Aryloxycarbonyl groups such as; benzenesulfonyl groups, arylsulfonyl groups such as tosyl groups; trityl groups, benzyl groups,
An aralkyl group such as a benzoyl group can be mentioned.
The “alkylamino group” is a lower alkylamino group such as methylamino and ethylamino.

Next, the production method of the novel 2-alkynylmalonic acid derivative [I] according to the present invention will be described with reference to general production methods and specific production examples. These derivatives [I]
Needless to say, the production of is not limited to the following method. For more details, it is advisable to refer to individual manufacturing examples.

[0010]

[Chemical 2]

(First Step) Production of Compound [3] This step itself includes aza-Witt
ig) Well known as the reaction (Tetrahed
ron Lett. , 22 (46), 4607 (198)
1)). The compound [1] is a diester of ketomalonic acid, and examples thereof include diethyl ketomalonic acid ester, dimethyl ketomalonic acid ester, dibenzyl ketomalonic acid ester, and diphenacetyl ketomalonic acid ester. The compound [2] is an acetyl group,
An iminotriphenylphosphine protected with an amino protecting group (R ^{2 ′} ) such as a butyryl group, a benzoyl group, a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Z), and a p-nitrobenzyloxycarbonyl group. And preferably acetyliminotriphenylphosphine. The compound [3] is prepared by dissolving the compound [2] in an organic solvent such as anhydrous tetrahydrofuran (THF) or anhydrous dioxane, and then adding 1 in the same solution.
It can be obtained by adding an equivalent amount of the compound [1] and reacting in an inert gas atmosphere for several hours to several tens of hours.

(Second Step) Production of Compound [I] The compound [I] is obtained by isolating the compound [3] obtained in the first step, or by directly isolating the compound [3] from the lithium acetylide [4] without isolation. -10 ° C to -80 ° C under active gas atmosphere
It is obtained by reacting under the conditions of and alkynylating. Lithium acetylide [4] is obtained by subjecting an acetylene derivative having a corresponding substituent R (R is as described above) to an exchange reaction with n-butyllithium in an organic solvent such as anhydrous THF, anhydrous ether or anhydrous dimethoxyethane. Can be easily obtained. When a compound [I] having a hydroxy-substituted lower alkyl group such as a 2-hydroxyethyl group as the R group is desired, a Li—C≡C—C ₂ H ₄ —O-protecting group (tetrahydro) is used as the compound [4]. Pyranyl group, methoxymethyl group, benzyl group, p-nitrobenzyl group, etc.)
This should be deprotected. Further, when the compound [I] in which R ² is an amino group is desired, it may be deprotected after preparing a compound such as R ^{2 ′} is Boc-according to the above method.

(Third Step) Production of Compound [I] (where R = -H) A compound [5] having a trimethylsilylethynyl group, for example, ethyl, according to the method of the first and second steps.
After obtaining 2-acetamido-2-ethoxycarbonyl-4-trimethylsilyl-3-butyric acid ester, the trimethylsilyl group is easily eliminated by desilylation to obtain the desired compound [I] in which R is -H. Can be obtained. Specifically, the compound [5] is dissolved in a non-reactive polar organic solvent such as THF, dioxane, and acetone,
The trimethylsilyl group can be eliminated by reacting in an inert gas atmosphere at 20 ° C. to −80 ° C., preferably −80 ° C. in the presence of 1 equivalent to 2 equivalents of an acid. Preferable acids include organic acids such as acetic acid, oxalic acid and citric acid, or inorganic acids such as hydrochloric acid. Acetic acid is particularly preferable, and in this case, good results can be obtained by additionally using a fluorine salt of a quaternary amine such as tetrabutylammonium fluoride.

(Step 4) Production of Compound [6] This method is effective when R ² in the general formula [I] is a hydroxyl group or a lower alkoxy group. The compound [6] is obtained by stirring and reacting the lithium compound [4] dissolved in an organic solvent such as tetrahydrofuran with the compound [1] at room temperature or under cooling, preferably in a nitrogen atmosphere.

(Step 5) Production of Compound [I] The target compound [I '] (wherein R ³ represents a hydrogen atom or a lower alkyl group) is the compound [6] obtained in the above Step 4. It is obtained by treatment with an acid such as hydrochloric acid, isolated or without isolation, or reaction with a dialkylsulfuric acid such as dimethylsulfuric acid dissolved in an organic solvent such as tetrahydrofuran.

The compounds represented by the general formula [I] in the present invention are, for example, as shown in Tables 1 and 2 below.

[0017]

[Table 1]

[0018]

[Table 2]

Next, a specific method for producing these compounds will be described. Production Example 1 Ethyl 2-acetylamino-2-ethoxycarbonyl-4-phenyl-3-butynoate (Compound 1) Acetylamino-triphenylphosphine (1.500 g, 4.
69 mmol) was dissolved in anhydrous tetrahydrofuran (40 ml),
Diethyl ketomalonate (0.7ml, 4.69mmol) at room temperature
Was added and stirred under a nitrogen atmosphere, and the resulting pale yellow solution was heated under reflux for 10 hours. Next, lithium phenylacetylide prepared by reacting phenylacetylene (0.515 mg, 4.69 mmol) with a hexane solution of n-butyllithium (2.80 ml, 4.69 mmol) was added to the above reaction solution at −78 ° C. under a nitrogen atmosphere. The mixture was stirred for 1 hour. After 3 hours, 5%
The reaction is terminated by adding hydrochloric acid (4 ml),
After warming the reaction solution to room temperature, ether (50 ml x 3)
The extract was extracted with and the ether layer was washed with saturated aqueous sodium hydrogen carbonate solution (40 ml) and saturated brine (40 ml), and dried over anhydrous magnesium sulfate. After filtration, it was distilled off under reduced pressure until it became a brown viscous liquid, which was subjected to column chromatography (ether-n-
Purification with hexane (5: 1) gave the title compound as a pale yellow oil (1.000 g, yield 68%). ¹ HNMR (200MHz, CDCl ₃ ) δ1.33 (6H, t, J = 7.1Hz), 2.10 (3H,
s), 4.36 (4H, q, J = 7.1Hz), 7.04 (1H, brs), 7.21-7.52 (5H,
m); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 13.88,22.80,60.72,63.71,82.4
7,84.70,121.87,128.15,128.86,132.16,165.08,168.73; IR (neat) 3362,2985,2239,1752,1673,1491cm ^-1 Theoretical value C ₁₇ H ₁₉ NO ₅ : C, 64.34; H, 6.03; N, 4.41, Measured value C, 6
3.88; H, 6.24; N, 4.28

Production Example 2 Ethyl 2-acetylamino-2-ethoxycarbonyl-4-p-toluyl-3-butynoate (Compound 2) p-Tolylacetylene was subjected to the same reaction as in Production Example 1,
The title compound was obtained as colorless needles. Melting point: 132-133 ° C ¹ H NMR (200MHz, CDCl ₃ ) δ1.32 (6H, t, J = 7.1Hz), 2.09 (3H,
s), 2.33 (3H, s), 4.35 (4H, q, J = 7.1Hz), 7.04 (1H, brs), 7.10
(2H, AB, J _AB = 8.3Hz), 7.37 (2H, AB, J _AB = 8.3Hz); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.89,21.53,22.85,60.73,63.6
8,81.74,84.96,118.79,128.89,132.08,139.03,165.18,1
68.68; IR (KBr) 3363,2985,2238,1757,1682,1505cm ^-1 Theoretical value C ₁₈ H ₂₁ NO ₅ : C, 65.24; H, 6.39; N, 4.23, Measured value C, 6
4.84; H, 6.56; N, 4.11

Production Example 3 Ethyl 2-acetylamino-2-ethoxycarbonyl-4- (1-cyclohexen-1-yl) -3-butynoate (Compound 3) 1-Cyclohexen-1-ylacetylene was produced in the same manner as in Production Example 1. To give the title compound as colorless needles. Melting point: 82 ° C ¹ H NMR (200MHz, CDCl ₃ ) δ1.30 (6H, t, J = 7.1Hz), 1.50-1.80
(4H, m), 2.06 (3H, s), 2.00-2.35 (3H, m), 4.32 (4H, q, J = 7.1H
z), 6.17-6.28 (1H, m), 6.93 (1H, brs) ,; ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.88,21.36,22.13,22.86,25.6
3,28.71,60.60,63.57,79.64,86.61,119.54,137.07,165.
29,168.54; IR (KBr) 3251,2215,1752,1656,1510cm ^-1 Theoretical value C ₁₇ H ₂₃ NO ₅ : C, 63.52; H, 7.22; N, 4.36, Measured value C, 6
3.39; H, 7.25; N, 4.12

Production Example 4 Ethyl 2-acetylamino-2-ethoxycarbonyl-3-heptinoate (Compound 4) 1-Pentine was subjected to the same reaction as in Production Example 1 to obtain a white powdery title compound. Melting point: 53-54 ° C ¹ H NMR (200MHz, CDCl ₃ ) δ 0.97 (3H, t, J = 7.3Hz), 1.30 (6H, t,
J = 7.1Hz), 1.48-1.60 (2H, m), 2.06 (3H, s), 4.31 (4H, q, J = 7.
1Hz), 6.94 (1H, brs) ,; ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.33,13.86,20.80,21.67,22.7
9,60.28,63.49,73.84,86.01,165.45,168.68; IR (neat) 3370,2968,2875,2247,1752,1668,1505,1370,12
68,1125,1016cm ^-1 Theoretical value C ₁₄ H ₂₁ NO ₅ : C, 59.33; H, 7.47; N, 4.95, Measured value C, 5
9.40; H, 7.54; N, 4.72

Production Example 5 Ethyl 2-acetylamino-2-ethoxycarbonyl-4-trimethylsilyl-3-butynoate (Compound 5) Trimethylsilylacetylene was subjected to the same reaction as in Production Example 1 to give the title compound as colorless needles. Obtained. Melting point: 97 ° C ¹ H NMR (200MHz, CDCl ₃ ) δ0.18 (9H, s), 1.30 (6H, t, J = 7.1H
z), 2.06 (3H, s), 4.31 (2H, q, J = 7.1Hz), 4.32 (2H, q, J = 7.1H
z), 6.88 (1H, brs) ,; ¹³ C NMR (100MHz, CDCl ₃ ) δ 0.34,14.13,23.14,61.00,63.9
1,90.84,97.85,165.22,168.82; IR (KBr) 3286,2986,2239,1752,1671,1490cm ^-1 Theoretical value C ₁₄ H ₂₃ NO ₅ Si: C, 53.65; H, 7.40; N, 4.47, measured Value C,
53.39; H, 7.53; N, 4.22

Production Example 6 Ethyl 2-acetylamino-2-ethoxycarbonyl-3-butynoate (Compound 6) The compound (0.157 mg, 0.5 mmol) obtained in Production Example 5 was dissolved in anhydrous tetrahydrofuran (12 ml) to prepare acetic acid. (0.057m
l, 1 mmol) and 1 ml of an anhydrous tetrahydrofuran solution of tetrabutylammonium fluoride (1 mmol, 1.0M) were added, and the mixture was stirred at -78 ° C for 3 hours under a nitrogen atmosphere. The reaction mixture was warmed to room temperature and extracted with ethyl acetate (40 ml x 3). The ethyl acetate layer was washed with water (20 ml) and saturated saline (20 ml).
After washing with, it was dehydrated with anhydrous sodium sulfate. After filtration, it was distilled off under reduced pressure until a brown residue was obtained and purified by column chromatography (ether-n-hexane (5: 1)) to give the title compound as a white solid (0.095 g, yield 80%). Got Melting point: 125 ° C ¹ H NMR (200MHz, CDCl ₃ ) δ1.31 (6H, t, J = 7.1Hz), 2.08 (3H,
s), 2.56 (1H, s), 4.34 (4H, q, J = 7.1Hz), 6.95 (1H, brs) ,; ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.82,22.72,60.03,63.93,73.3
1,76.92,164.56,168.87; IR (neat) 3282,2120,1769,1659,1515cm ^-1 Theoretical value C ₁₁ H ₁₅ NO ₅ : C, 54.75; H, 6.27; N, 5.81, Measured value C, 5
4.41, H; 6.30; N, 5.73

Production Example 7 Ethyl 2-benzoylamino-2-ethoxycarbonyl-4-p-toluyl-3-butynoate (Compound 7) Benzoyliminotriphenylphosphine (0.381 g, 1 m
mol) was dissolved in anhydrous tetrahydrofuran (10 ml), diethyl ketomalonate (0.15 ml, 1 mmol) was added at room temperature, and the mixture was stirred under a nitrogen atmosphere. The resulting pale yellow solution is 20
After heating under reflux for an hour, it was cooled to -78 ° C. Next, p-tolylacetylene (0.15 ml, 1.2 mmol) and n-butyllithium (1.61 M) in hexane were added to this solution (0.75 m
1, 1.2 mmol) was reacted at 0 ° C. for 30 minutes, and a solution of lithium-p-tolyl acetylide in anhydrous tetrahydrofuran (5 ml) was added. After 3 hours, the reaction was terminated by adding 5% hydrochloric acid (4 ml), the reaction solution was warmed to room temperature, and then diluted with ether (50 ml). The obtained ether layer was washed with water (15 ml), saturated aqueous sodium hydrogen carbonate solution (15 ml) and saturated brine (15 ml), and dried over anhydrous magnesium sulfate. Concentration under reduced pressure gave a yellow oil (0.67
9 g) was purified by column chromatography (ether-n-hexane (2: 1)) to give the title compound (0.0
42 g, yield 11%) was obtained. ¹ HNMR (200MHz, CDCl ₃ ) δ1.34 (6H, t, J = 7.1Hz), 2.36 (3H,
s), 4.39 (4H, q, J = 7.1Hz), 7.07 (2H, AB, J _AB = 8.3Hz), 7.36 (2
H, AB, J _AB = 8.3Hz), 7.41-7.54 (3H, m), 7.69 (1H, s), 7.86-7.
90 (2H, m); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.92,21.52,61.01,63.75,77.2
2,85.16,118.83,127.38,128.62,128.86,132.11,132.99,
139.00,165.26,165.56; IR (neat) 3418,2982,2238,1751,1674,1603,1581,1509,14
73,1369,1269,1207,1097,1070cm ^-1 HRMS theoretical value C ₂₃ H ₂₃ NO ₅ MW: 393.1576, measured value m / z: 39
3.1571

Production Example 8 Ethyl 2-butanoylamino-2-ethoxycarbonyl-4-p-toluyl-3-butynoate (Compound 8) Butanoyliminotriphenylphosphine (0.521 g, 1.
5 mmol) in anhydrous tetrahydrofuran (15 ml),
Diethyl ketomalonate (0.23 ml, 1.5 mmol) at room temperature
Was added and the mixture was stirred under a nitrogen atmosphere. The resulting pale yellow solution was heated under reflux for 12 hours and then cooled to -78 ° C. Then p-tolyl acetylene (0.23 ml, 1.8 mmo
l) and n-butyllithium (1.61M) in hexane (1.
12 ml (1.8 mmol) was reacted at 0 ° C. for 30 minutes, and a solution of lithium-p-tolylacetylide in anhydrous tetrahydrofuran (10 ml) was added. After 3 hours, 5% hydrochloric acid (4m
The reaction was terminated by adding l), and the reaction solution was warmed to room temperature and then diluted with ether (80 ml).
The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated brine (20 ml), and dried over anhydrous magnesium sulfate. The yellow oil (0.902 g) obtained by concentrating under reduced pressure together with crystals of triphenylphosphine oxide was subjected to column chromatography (ether-n
-Hexane (2: 1)) to give the title compound (0.291 g, yield 54%) as a white solid. Melting point: 60.5 to 61 ° C ¹ H NMR (200MHz, CDCl ₃ ) δ 0.98 (3H, t, J = 7.3Hz), 1.32 (6H, t,
J = 7.1Hz), 1.56-1.58 (2H, m), 2.28 (2H, t, J = 7.3Hz), 2.34 (3
H, s), 4.35 (4H, q, J = 7.1Hz), 6.98 (1H, s), 7.08 (2H, AB, J _AB =
8.1Hz), 7.34 (2H, AB, J _AB = 8.1Hz); ¹³ CNMR (100MHz, CDCl ₃ ) δ 13.54,13.88,18.89,21.52,37.8
2,38.14,60.65,63.61,81.94,84.82,118.89,128.89,132.
05,138.98,165.22,171.57; IR (KBr) 3387,3034,2973,2872,2236,1911,1763,1688,148
4,1389,1364,1287,1161,1097,1044,1016cm ^-1 HRMS theoretical value C ₂₀ H ₂₅ NO ₅ MW: 359.1733, measured value m / z: 35
9.1753

Production Example 9 Ethyl 2-methoxy-2-ethoxycarbonyl-4-
p-Toluyl-3-butynoate (Compound 9) Diethylketomalonate (0.76 ml, 5 mmol) in anhydrous tetrahydrofuran (10 ml) was charged with p-tolylacetylene (0.63 ml, 5 mmol) and n-butyllithium (1.63M). A solution of lithium-p-tolyl-acetylide in anhydrous tetrahydrofuran (20 ml) prepared by reacting a hexane solution (3.07 ml, 5 mmol) at 0 ° C. for 30 minutes under a nitrogen atmosphere
It was added dropwise at 78 ° C. 2 hours later, dimethyl sulfate (0.49m
(1, 5 mmol) was added dropwise, the reaction solution was warmed to room temperature over 1 hour, and then heated to reflux. After 3.5 hours, the reaction was cooled to room temperature and diluted with ether (80 ml). The obtained ether layer was washed with water (30 ml), saturated aqueous sodium hydrogen carbonate solution (30 ml) and saturated brine (30 ml), and dried over anhydrous magnesium sulfate. The brown oil (1.4 g) obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (2: 3)) to give the title compound as a white solid (0.942 g, yield 62%). Got ¹ H NMR (200MHz, CDCl ₃ ) δ1.33 (6H, t, J = 7.1Hz), 2.36 (3H,
s), 3.58 (3H, s), 4.34 (4H, q, J = 7.1Hz), 7.12 (2H, AB, J _AB = 8.
3Hz), 7.39 (2H, AB, J _AB = 8.3Hz); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.99,21.54,54.37,62.97,79.8
7,80.08,89.52,118.30,129.10,132.07,139.58,165.40; IR (neat) 2984,2232,1752,1510,1447,1368,1255,1109,10
35cm ^-1 HRMS Theoretical C ₁₇ H ₂₀ O ₅ MW: 304.1311, Measured m / z: 30
4.1295

Production Example 10 Ethyl 2-methoxy-2-ethoxycarbonyl-4-
Phenyl-3-butynoate (Compound 10) Diethyl ketomalonate (0.46 ml, 3 mmol) in anhydrous tetrahydrofuran (10 ml) was added to phenylacetylene (0.33 ml, 3 mmol) and n-butyllithium (1.63M) in hexane (1.84). ml, 3 mmol) was reacted at 0 ° C. for 30 minutes to prepare a solution of lithium-phenylacetylide in anhydrous tetrahydrofuran (15 ml) under a nitrogen atmosphere at −78.
It was added dropwise at ° C. 2 hours later, dimethylsulfate (0.28ml, 3m
(mol) was added dropwise, the reaction solution was warmed to room temperature over 1 hour, and then heated to reflux. After 3.5 hours, the reaction was cooled to room temperature and diluted with ether (80 ml). The obtained ether layer was washed with water (30 ml), saturated aqueous sodium hydrogen carbonate solution (30 ml) and saturated brine (30 ml), and dried over anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was subjected to column chromatography (ether-n-hexane (2:
After purification in 3)), the title compound (0.454 g, yield 52%) was obtained as a yellow oil. ¹ HNMR (200MHz, CDCl ₃ ) δ1.34 (6H, t, J = 7.1Hz), 3.60 (3H,
s), 4.35 (4H, q, J = 7.1Hz), 7.27-7.38 (3H, m), 7.50-7.55 (2
H, m); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 14.12,54.44,62.98,80.06,80.5
8,89.31,121.42,128.39,129.35,132.20,165.35; IR (neat) 2984,2833,2233,1752,1491,1446,1391,1369,12
55,1110,1034cm ^-1 HRMS Theoretical C ₁₆ H ₁₈ O ₅ MW: 290.1154, Measured m / z: 29
0.1158

Production Example 11 Ethyl 2-methoxy-2-ethoxycarbonyl-4-
(1-Cyclohexen-1-yl) -3-butynoate (Compound 11) Diethyl ketomalonate (0.763 ml, 5 mmol) in anhydrous tetrahydrofuran (10 ml) was added with 1-cyclohexene-
A solution of 1-ylacetylene (0.59 ml, 5 mmol) and n-butyllithium (1.61M) in hexane (3.11 ml, 5 mmol) was added to 0.
Lithium-1-cyclohexenyl acetylide anhydrous tetrahydrofuran (20m
l) The solution was added dropwise at -78 ° C under a nitrogen atmosphere. After 2 hours, dimethylsulfate (0.47 ml, 5 mmol) was added dropwise,
The reaction solution was heated to room temperature over 1 hour and then heated to reflux. After 3.5 hours, the reaction was cooled to room temperature and diluted with ether (80 ml). The ether layer obtained was washed with water (30 m
l), saturated aqueous sodium hydrogen carbonate solution (30 ml) and saturated saline (30 ml), and then dried over anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (2: 3)) to give the title compound (0.911 g, yield 62%) as a yellow oil. ¹ H NMR (200MHz, CDCl ₃ ) δ1.31 (6H, t, J = 7.1Hz), 1.46-
1.72 (4H, m), 1.97-2.21 (4H, m), 3.51 (3H, s), 4.31 (4H, q, J =
7.1Hz), 6.20-6.32 (1H, m); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.98, 14.02, 21.35, 22.12, 25.7
0,28.71,54.23,62.80,77.74,80.02,91.31,119.38,137.7
3,165.57; IR (neat) 2937,2220,1752,1448,1391,1368,1222,1109,10
39cm ^-1 HRMS Theoretical C ₁₆ H ₂₂ O ₅ MW: 294.1467, Measured m / z: 29
4.1475 Theoretical C ₁₆ H ₂₂ O ₅ : C, 65.29; H, 7.53, Measured C, 64.82; H, 7.
73

Production Example 12 Ethyl 2-methoxy-2-ethoxycarbonyl-3-
Heptinoate (Compound 12) 1-pentyne (0.49m) was added to a solution of diethyl ketomalonate (0.76ml, 5mmol) in anhydrous tetrahydrofuran (10ml).
l, 5 mmol) and n-butyllithium (1.61M) in hexane (3.11 ml, 5 mmol) were reacted at 0 ° C. for 30 minutes to prepare a solution of lithium-propylacetylide in anhydrous tetrahydrofuran (20 ml) under a nitrogen atmosphere. It was added dropwise at 78 ° C. After 2 hours, dimethylsulfate (0.47 ml, 5 mmol) was added dropwise, the reaction solution was warmed to room temperature over 1 hour, and then heated to reflux. After 3.5 hours, the reaction was cooled to room temperature and diluted with ether (80 ml). The ether layer obtained was washed with water (30 ml) and saturated aqueous sodium hydrogen carbonate solution (30 m
After washing with l) and saturated saline (30 ml), it was dehydrated with anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (1: 1)) to give the title compound as a yellow oil (0.521 g, yield 41).
%) Was obtained. ¹ HNMR (200MHz, CDCl ₃ ) δ1.01 (3H, t, J = 7.3Hz), 1.30 (6H, t,
J = 7.1Hz), 1.50-1.72 (2H, m), 2.30 (2H, t, J = 7.0Hz), 3.50 (3
H, s), 4.30 (4H, q, J = 7.1Hz); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.39,13.98,20.82,21.67,54.1
0,62.80,71.96,79.67,90.92,165.70; IR (neat) 2967,2833,2242,1752,1465,1368,1269,1109,10
44 cm ^-1 Theoretical value C ₁₃ H ₂₀ O ₅ : C, 60.91; H, 7.87, measured value C, 60.61; H, 8.
07

Production Example 13 Ethyl 2-hydroxy-2-ethoxycarbonyl-4
-P-Toluyl-3-butynoate (Compound 13) Diethylketomalonate (0.46 ml, 3 mmol) in anhydrous tetrahydrofuran (10 ml) was added with p-tolylacetylene (0.46 ml, 3 mmol) and n-butyllithium (1.63M). A solution of lithium-p-tolylacetylide in anhydrous tetrahydrofuran (15 ml) prepared by reacting hexane solution (1.84 ml, 3 mmol) of the above with 30 minutes at 0 ° C. under a nitrogen atmosphere at −7.
It was added dropwise at 8 ° C. After 2 hours, the reaction was terminated by adding 5% hydrochloric acid (4 ml), the reaction solution was warmed to room temperature, and diluted with ether (50 ml). The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated brine (20 ml), and dried over anhydrous magnesium sulfate. The yellow oil (0.893 g) obtained by concentration under reduced pressure was subjected to column chromatography (ether-n-
Purification with hexane (1: 1) gave the title compound (0.635 g, yield 73%) as a yellow solid. ¹ H NMR (200MHz, CDCl ₃ ) δ1.34 (6H, t, J = 7.1Hz), 2.35 (3H,
s), 4.20 (1H, s), 4.36 (4H, q, J = 7.2Hz), 7.11 (2H, AB, J _AB = 8.
1Hz), 7.36 (2H, AB, J _AB = 8.1Hz); ¹³ CNMR (100MHz, CDCl ₃ ) δ13.88,21.47,63.59,66.74,72.9
6,81.94,118.35,129.96,131.95,139.34,167.26; IR (KBr) 3467,2983,2238,1757,1511,1450,1369,1264,122
8,1104,1064,1026cm ^-1 HRMS theoretical value C ₁₆ H ₁₈ O ₅ MW: 290.1154, measured value m / z: 29
0.1151

Production Example 14 Ethyl 2-hydroxy-2-ethoxycarbonyl-4
-Phenyl-3-butynoate (Compound 14) Diethyl ketomalonate (0.46 ml, 3 mmol) in anhydrous tetrahydrofuran (10 ml) was added to phenylacetylene (0.33 ml, 3 mmol) and n-butyllithium (1.63M) in hexane ( 1.84 ml, 3 mmol) was reacted at 0 ° C. for 30 minutes to prepare a solution of lithium-phenylacetylide in anhydrous tetrahydrofuran (15 ml) under a nitrogen atmosphere at −78 ° C.
It was dripped at. After 2 hours, the reaction was terminated by adding 5% hydrochloric acid (4 ml), the reaction solution was warmed to room temperature, and diluted with ether (50 ml). The ether layer obtained was washed with water (20 ml) and saturated aqueous sodium hydrogen carbonate solution (20 m
After washing with l) and saturated saline (20 ml), it was dehydrated with anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (1: 1)) to give the title compound as a yellow oil (0.572 g, yield 69).
%) Was obtained. ¹ H NMR (200MHz, CDCl ₃ ) δ1.34 (6H, t, J = 7.1Hz), 4.23 (1H,
s), 4.36 (4H, q, J = 7.1Hz), 7.26-7.35 (3H, m), 7.46-7.52 (2
H, m); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 13.89,63.65,72.94,82.64,86.5
0,121.43,128.22,129.11,132.05,167.19; IR (KBr) 3468,2985,2238,1747,1491,1445,1369,1216,110
7,1071,1024cm ^-1 HRMS Theoretical C ₁₅ H ₁₆ O ₅ MW: 276.0998, Measured m / z: 27
6.0979 Theoretical C ₁₅ H ₁₆ O ₅ : C, 65.21; H, 5.84, Measured C, 64.88; H, 5.
98

Production Example 15 Ethyl 2-hydroxy-2-ethoxycarbonyl-4
-(1-Cyclohexen-1-yl) -3-butynoate (Compound 15) To a solution of diethyl ketomalonate (0.15 ml, 1 mmol) in anhydrous tetrahydrofuran (10 ml), 1-cyclohexene-
A solution of 1-ylacetylene (0.12 ml, 1 mmol) and n-butyllithium (1.63M) in hexane (0.61 ml, 1 mmol) was added to 0.
Lithium-1-cyclohexylacetylide anhydrous tetrahydrofuran (5 ml) prepared by reacting at 30 ° C for 30 minutes
The solution was added dropwise at -78 ° C under a nitrogen atmosphere. After 2 hours, the reaction was terminated by adding 5% hydrochloric acid (4 ml), and the temperature of the reaction solution was raised to room temperature.
l) diluted. The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated saline (20 ml).
(ml) and then dehydrated with anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (1: 1)) to give the title compound (0.188 g, yield 67%) as a yellow oil. ¹ H NMR (200MHz, CDCl ₃ ) δ1.32 (6H, t, J = 7.1Hz), 1.43-1.72
(4H, m), 2.05-2.18 (4H, m), 4.09 (1H, s), 4.33 (4H, q, J = 7.1H
z), 6.20-6.26 (1H, m); ¹³ CNMR (100MHz, CDCl ₃ ) δ 13.92,21.35,22.12,25.67,28.6
1,63.51,72.96,80.05,88.51,119.39,137.56,165.45; IR (neat) 3474,2937,2225,1747,1448,1369,1207,1098,10
29cm ^-1 HRMS Theoretical C ₁₅ H ₂₀ O ₅ MW: 280.1311, Measured m / z: 28
0.1300 theoretical value C ₁₅ H ₂₀ O ₅ : C, 64.26; H, 7.20, measured value C, 64.38; H, 7.
40

Production Example 16 Ethyl 2-hydroxy-2-ethoxycarbonyl-3
-Heptinoate (Compound 16) To a solution of diethyl ketomalonate (0.15 ml, 1 mmol) in anhydrous tetrahydrofuran (10 ml), 1-pentyne (0.1 m
l, 1 mmol) and n-butyllithium (1.63M) in hexane (0.61 ml, 1 mmol) were reacted at 0 ° C. for 30 minutes to prepare a solution of lithium-propylacetylide in anhydrous tetrahydrofuran (5 ml) under a nitrogen atmosphere. It was added dropwise at 78 ° C. After 2 hours, the reaction was terminated by adding 5% hydrochloric acid (4 ml), the reaction solution was warmed to room temperature, and diluted with ether (50 ml). The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated brine (20 ml), and dried over anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (1: 1)) to give the title compound (0.133 g, yield 55%) as a yellow oil. ¹ H NMR (200MHz, CDCl ₃ ) δ 1.00 (3H, t, J = 7.3Hz), 1.32 (6H, t,
J = 7.1Hz), 2.00-2.20 (2H, m), 2.26 (2H, t, J = 7.1Hz), 4.04 (1
H, s), 4.33 (4H, q, J = 7.1Hz); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.35,13.92,20.75,21.57,63.4
7,72.62,74.29,88.12,165.59; IR (neat) 3475,2967,2245,1747,1466,1369,1269,1176,10
94,1028 cm ^-1 theoretical value C ₁₂ H ₁₈ O ₅ : C, 59.48, H, 7.49, measured value C, 59.13; H, 7.
63

Production Example 17 Ethyl 2-hydroxy-2-ethoxycarbonyl-4
-Trimethylsilyl-3-butynoate (Compound 17) To a solution of diethyl ketomalonate (0.46 ml, 3 mmol) in anhydrous tetrahydrofuran (10 ml) was added trimethylsilylacetylene (0.42 ml, 1 mmol) and n-butyllithium (1.63).
A solution of lithium-trimethylsilylacetylide in anhydrous tetrahydrofuran (15 ml) prepared by reacting a solution of M) in hexane (1.84 ml, 3 mmol) at 0 ° C. for 30 minutes was added dropwise at −78 ° C. under a nitrogen atmosphere. After 2 hours, 5% hydrochloric acid (4m
The reaction was terminated by adding l), the reaction was warmed to room temperature and diluted with ether (50 ml). The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated brine (20 ml), and dried over anhydrous magnesium sulfate. The residue obtained by concentration under reduced pressure was purified by column chromatography (ether-n-hexane (1: 1)) to give the title compound (0.512) as a yellow oil.
g, yield 63%). ¹ H NMR (200MHz, CDCl ₃ ) δ0.20 (9H, s), 1.32 (6H, t, J = 7.1H
z), 4.08 (1H, s), 4.33 (4H, q, J = 7.1Hz); ¹³ C NMR (100MHz, CDCl ₃ ) δ-0.50,13.81,63.54,72.72,92.5
6,97.90,166.97; IR (neat) 3475,2965,2117,1747,1369,1252,1129,1082,10
25cm ^-1 HRMS Theoretical C ₁₂ H ₂₀ O ₅ Si MW: 272.1080, Measured m / z: 2
72.1100 Theoretical C ₁₂ H ₂₀ O ₅ Si: C, 52.92; H, 7.41, Measured C, 52.69; H,
7.61

Production Example 18 Ethyl 2- (N-acetyl-N-methylamino) -2
-Ethoxycarbonyl-4-p-toluyl-3-butynoate (Compound 18) Acetylamino-triphenyl-phosphine (0.320 g,
1.0 mmol) is dissolved in anhydrous tetrahydrofuran (8 ml),
Diethyl ketomalonate (0.16 ml, 1.0 mmol) at room temperature
Was added and the mixture was stirred under a nitrogen atmosphere. The resulting pale yellow solution was heated under reflux for 12 hours and then cooled to -78 ° C. This solution was then added to p-tolyl acetylene (0.13 ml, 1.0 mmo
l) and n-butyllithium (1.61M) in hexane (0.
65 ml (1.0 mmol) was reacted at 0 ° C. for 30 minutes, and a solution of lithium-p-tolylacetylide in anhydrous tetrahydrofuran (10 ml) was added. After 3 hours, dimethylsulfate (0.01 ml, 1 mmol) was added dropwise, and the reaction mixture was allowed to reach room temperature until 1 hour.
The temperature was raised for a time, and then the mixture was heated under reflux. After 3 hours, the reaction was cooled to room temperature and diluted with ether (50 ml). The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated brine (20 ml), and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the obtained yellow oil (0.694 g) was purified by column chromatography (ether-n-hexane (2: 1)) to give the title compound (0.064 g, yield 19%) as a yellow oil. ) And ethyl 2-acetylamino-2-ethoxycarbonyl-4-p-toluyl-3-butynoate (0.100 g, yield 30%). ¹ H NMR (200MHz, CDCl ₃ ) δ1.33 (6H, t, J = 7.1Hz), 2.20 (3H,
s), 2.36 (3H, s), 3.24 (3H, s), 4.22-4.48 (4H, m), 7.12 (2H, A
B, J _AB = 8.3Hz), 7.35 (2H, AB, J _AB = 8.3Hz); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.96,21.56,22.48,34.55,62.9
3,80.69,88.91,118.30,129.07,131.87,139.61,164.66,1
71.79; IR (neat) 2984,2235,1751,1668,1511,1447,1387,1346,12
12,1083,1047cm ^-1 HRMS Theoretical C ₁₉ H ₂₃ NO ₅ MW: 345.1576, Measured m / z: 34
5.1577

Production Example 19 Ethyl 2- (N-acetyl-N-methylamino) -2
-Ethoxycarbonyl-4- (1-cyclohexene-1)
-Yl) -3-butynoate (Compound 19) Acetylimino-triphenyl-phosphine (1.597 g,
5 mmol) was dissolved in anhydrous tetrahydrofuran (8 ml), diethyl ketomalonate (0.763 ml, 5 mmol) was added at room temperature, and the mixture was stirred under a nitrogen atmosphere. The resulting pale yellow solution is 12
After heating under reflux for an hour, it was cooled to -78 ° C. This solution was then added to 1-cyclohexen-1-ylacetylene (0.
71 ml, 6 mmol) and a hexane solution (3.73 ml, 5 mmol) of n-butyllithium (1.61M) were reacted at 0 ° C. for 30 minutes to prepare a solution of lithium-1-cyclohexenylacetylide in anhydrous tetrahydrofuran (20 ml). . After 3 hours, dimethylsulfate (0.47 ml, 5 mmol) was added dropwise, the reaction solution was warmed to room temperature over 1 hour, and then heated to reflux. After 3 hours, the reaction solution was cooled to room temperature and washed with ether (50
ml). The obtained ether layer was washed with water (20 ml), saturated aqueous sodium hydrogen carbonate solution (20 ml) and saturated saline (20 ml).
(ml) and then dehydrated with anhydrous magnesium sulfate. After concentration under reduced pressure, the obtained yellow oil (0.694 g) was purified by column chromatography (ether-n-hexane (5: 1)) to give the title compound as a yellow oil (0.898 g, yield 54%). ) Got. ¹ H NMR (200MHz, CDCl ₃ ) δ1.31 (6H, t, J = 7.1Hz), 1.51-1.74
(4H, m), 2.00-2.14 (4H, m), 2.17 (3H, s), 3.17 (3H, s), 4.14-
4.43 (4H, m), 6.14-6.29 (1H, m); ¹³ C NMR (100MHz, CDCl ₃ ) δ 13.95,21.31,22.06,22.48,25.6
6,28.58,34.43,62.79,67.70,78.65,90.61,119.40,137.5
1,164.82,171.71; IR (neat) 2932,2223,1749,1668,1447,1387,1347,1207,10
49cm ^-1 HRMS Theoretical value C ₁₈ H ₂₅ NO ₅ MW: 335.1733 Measured value m / z: 33
5.1724

Production Example 20 Ethyl 2-acetylamino-2-ethoxycarbonyl-6- (tetrahydropyran-2-yloxy) -3-
Hexinoate (Compound 20) Acetyliminotriphenylphosphine (2.00 g, 6.2
36 mmol) in anhydrous tetrahydrofuran (30 ml),
Diethyl ketomalonate (1.05ml, 0.689mmo at room temperature)
l) was added, and the mixture was heated under reflux for 11 hours in a nitrogen atmosphere. Then 2- (3-butynyloxy) -tetrahydro-2H-pyran (1.16 g, 7.522 mmol) was dissolved in anhydrous tetrahydrofuran (30 ml) and n-butyllithium (1.64M) in hexane solution (4.58 ml, 7.522 mmol) was added. In addition, under ice cooling 30
The lithio compound solution prepared by stirring for 1 minute was added to the above reaction solution.
The mixture was added dropwise at 78 ° C and stirred for 3 hours. 5% Hydrochloric acid was added to the reaction solution, extracted with ether, washed with a saturated aqueous solution of sodium hydrogencarbonate, and then treated by a conventional method to purify the resulting residue by column chromatography (ether), which was yellow and transparent. The title compound as an oily substance (1.70 g, yield 74
%) Was obtained. ¹ HNMR (200MHz, CDCl ₃ ) δ1.29 (6H, t, J = 7.1Hz), 1.40-1.90
(6H, m), 2.05 (3H, s), 2.56 (2H, t, J = 7.3Hz), 3.45-3.65 (2H,
m), 3.75-3.95 (2H, m), 4.25-4.40 (4H, m), 4.60-4.70 (1H,
m), 6.92 (1H, brs); IR (neat) 2942,2254,1752,1687,1500,1369,1200,1123,10
34 cm ^-1

Production Example 21 Ethyl 2-acetylamino-2-ethoxycarbonyl-6-hydroxy-3-hexinoate (Compound 21) The compound obtained in Production Example 20 was mixed with acetic acid-tetrahydrofuran-water (4: 2: 1). After dissolving in a solvent (6 ml), 45 ℃
It was heated to and stirred for 3 hours. The reaction solution was extracted with ethyl acetate, washed with a saturated aqueous solution of sodium hydrogen carbonate, and then treated by a conventional method to purify the resulting residue by column chromatography (ether), and the title compound was obtained as a colorless transparent oily substance. (69.4 mg, yield 90%) was obtained. ¹ H NMR (200MHz, CDCl ₃ ) δ1.30 (6H, t, J = 7.1Hz), 2.06 (3H,
s), 2.50 (2H, t, J = 6.1Hz), 2.60-2.80 (1H, brs), 3.73 (2H, t,
J = 6.1Hz), 4.32 (4H, q, J = 7.1Hz), 7.06 (1H, brs); IR (neat) 3650-2600,2254,1751,1667,1500,1370,1196,11
26cm ^-1 HRMS Theoretical C ₁₃ H ₁₉ NO ₆ MW: 285.1212, Measured m / e: 28
5.1200 (M ⁺ )

Production Example 22 Ethyl 2-acetylamino-6- (5-dimethylamino-1-naphthalenesulfonyloxy) -2-ethoxycarbonyl-3-hexinoate (Compound 22) The compound obtained in Production Example 21 (44.6 mg , 0.516 mmol) in anhydrous tetrahydrofuran (2 ml), and a solution of methyllithium in (1.14M) ether (137.1 μl, 0.171 mmol).
Was added and stirred at −78 ° C. for 30 minutes under a nitrogen atmosphere,
Dansyl chloride (50.6 mg, 0.187 mmol) was added, and the mixture was stirred at room temperature for 3 hours. A saturated ammonium chloride aqueous solution was added to the reaction solution, and the mixture was extracted with ether, and then treated by a conventional method to purify the resulting residue by column chromatography (ether), and the title compound (33.5 mg, a yellow transparent oily substance, Yield 41%) was obtained. ¹ H NMR (200 MHz, CDCl ₃ ) δ 1.26 (6 H, t, J = 7.1 Hz), 2.01 (3 H,
s), 2.61 (2H, t, J = 7.4Hz), 2.90 (3H, s), 4.06 (2H, t, J = 7.4H
z), 4.20-4.35 (4H, m), 6.87 (1H, brs), 7.22 (1H, d, J = 7.6H
z), 7.45-7.65 (2H, m), 8.15-8.30 (2H, m), 8.62 (1H, d, J = 8.5
Hz); IR (neat) 3416,3016,2261,1751,1687,1482,1361,1272,11
75,1013,981cm ^-1 HRMS Theoretical C ₂₅ H ₃₀ N ₂ O ₈ S MW: 518.1722, Measured m / e: 51
8.1701 (M ⁺ ) Theoretical value C ₂₅ H ₃₀ N ₂ O ₈ S: C, 57.90; H, 5.83; N, 5.40, Measured value C,
57.70; H, 5.90; N, 5.16

Production Example 23 Ethyl 2-t-butoxycarboxamide-2-ethoxycarbonyl-3-heptinoate (Compound 23) t-Butoxycarbonyliminotriphenylphosphine was treated in the same manner as in Production Example 20 to give a yellow transparent oil. To give the title compound (yield 77%). ¹ H NMR (200 MHz, CDCl ₃ ) δ 0.98 (3 H, t, J = 7.3 Hz), 1.29 (6 H, t,
J = 7.1Hz), 1.45 (9H, s), 1.50-1.70 (2H, m), 2.23 (2H, t, J = 7.
1Hz), 4.15-4.45 (4H, m), 5.90-7.05 (1H, brs); IR (neat) 3435,2979,2937,2246,1724,1477,1393,1368,12
56,1168,1023cm ^-1 HRMS Theoretical C ₁₇ H ₂₇ NO ₆ MW: 341.1916 (M ⁺ + H), Measured m /
e: 341.1919 (M ⁺ + H)

Production Example 24 Ethyl 2-amino-2-ethoxycarbonyl-3-heptinoate (Compound 24) The compound (2.08 g, 6.092 mmol) obtained in Production Example 23 was mixed with 4 parts.
A solution of N hydrochloric acid in dioxane (30 ml) was added, and the mixture was stirred under ice-cooling for 10 minutes and then at room temperature for 1 hour, and n-hexane was added thereto, and the resulting precipitate was repeatedly washed with n-hexane. Further purification by column chromatography (methylene chloride-methanol (20: 1)) gave the title compound (1.40 g, yield 95%) as a yellow transparent oil. ¹ H NMR (200MHz, CDCl ₃ ) δ0.99 (3H, t, J = 7.3Hz), 1.30 (6H, t,
J = 7.1Hz), 1.45-1.70 (2H, m), 2.23 (2H, t, J = 7.1Hz), 2.15-
2.35 (2H, brs), 4.28 (4H, q, J = 7.1Hz); IR (neat) 3393,3324,2967,2938,2254,1747,1585,1239,10
96,1028cm ^-1 FRBMS Theoretical C ₁₂ H ₁₉ NO ₄ MW: 242 (M ⁺ + H), Measured m / e: 242
(M ⁺ + H)

The thus-obtained 2-alkynylmalonic acid derivative-containing pharmaceutical composition can be prepared as a solid preparation for oral administration, a liquid preparation, an injection for parenteral administration, or a suppository by a conventional formulation method. It can be made into various preparations such as agents. The dose varies depending on the age, body weight, symptoms, therapeutic effect, and administration method, but is usually once to several times a day or once a few days to once a week with a period of 0.1 mg per adult.
It is orally administered in the range of 500 mg, or parenterally in the range of 0.02 mg to 100 mg per adult. In solid compositions for oral administration, one or more active substances are used in admixture with at least one inert diluent, optionally with excipients, binders, lubricants, disintegrants. It may also contain agents, solubilizers, stabilizers and the like. The tablets or pills may optionally be coated with a gastric or enteric substance film. Capsules include hard capsules and soft capsules. Liquid compositions for oral administration include solutions, emulsions, suspensions,
Includes syrups and elixirs. Such a liquid composition contains an inert diluent which is generally used, and additionally, an auxiliary agent such as a moistening agent and a suspending agent, a sweetening agent, a flavoring agent, an aromatic agent, an antiseptic agent. May be contained. Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. In such an injection, one or more active substances are used by mixing with at least one inert aqueous diluent or inert non-aqueous diluent, and if necessary, further preserved. Auxiliary agents such as agents, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents may be contained.
These are usually filtered (bacterial retention filter, etc.),
It is sterilized by blending a bactericide or gamma irradiation, or after these treatments, it is made into a solid composition by a method such as lyophilization, and sterile water or a sterile diluent for injection is added immediately before use. used. Alternatively, it may be used as it is in a liquid state. Next, more specific formulation methods, test methods and the results thereof will be described with reference to Examples.

[0046]

【Example】

1. Formulation example The powders having the above formulations were mixed, passed through a 60-mesh sieve, and then 280 mg of this powder was put into a No. 3 gelatin capsule to obtain a capsule. 52 mg of the compound, 49 mg of microcrystalline cellulose and a part of corn starch were mixed and granulated by a conventional method. After passing the obtained granular material through a sieve, the remaining corn starch and magnesium stearate were added and mixed. Then, the obtained granules are mixed with a tableting machine to obtain 15
The tablets were 0 mg. The drug is dissolved in distilled water for injection at the above blending ratio, and dust removal, sterilization, and filtration are performed. The filtered drug solution was filled in an ampoule that had been washed, dried and sterilized to obtain an injection.

[0047]

[Test Example] Next, the following test was conducted to demonstrate that the compound of the present invention suppresses the production of IL-1. Test Example 1 (1) Measurement of IL-1 production inhibitory activity from human peripheral monocytes Human venous blood was collected, and Lyphoprep ^™ (NYCOMED PHARMA AS
Liquid) was added to the mixture to obtain mononuclear cells by centrifugal separation under superficial gravity.
RPMI 16 containing 10% fetal calf serum (FCS)
After dispersing in 40 culture medium, culture 24-well plate (BECTON DI
(KINSON) was seeded at 5 × 10 ⁵ cells / ml / well and cultured at 37 ° C. for 2 to 3 hours in a 5% CO ² atmosphere. Thereafter, a DMSO solution containing a 2-alkynylmalonic acid derivative compound according to the present invention was added (DMSO final concentration: 0.5%), and 1 hour later, lipopolysaccharide (LPS: DIFCO) (final concentration: 30 μg / ml) was added. Plus 48
Incubated for hours. Thereafter, the culture supernatant was stored at -80 ° C until the IL-1 activity was measured. IL-1 activity is measured by I
Cell growth inhibitory factor (Growth Inhibition) using A375 cells (human melanoma cell line) whose growth is inhibited in the presence of L-1
Factor: GIF) activity (inflammation, see 8 (5) 409). That is, A375 cells were seeded at 2 × 10 ³ cells / 100 μl / well, and 5%
After culturing at 37 ° C. for 3 hours in a CO ² atmosphere, 100 μl / well of diluted monocyte cell supernatant was added and culturing was continued for 4 days. The cell proliferation was measured by fixing the cells with 100 μl / well of methyl alcohol, staining with 0.2% crystal violet for 10 minutes, washing and solubilizing the cells with 1% SDS, and then measuring the absorption wavelength at 550 nm. Further, the IL-1 amount was calculated from the standard curve obtained by adding the standard human IL-1β to the same cultured cells, and the IC ₅₀ was determined. Table 3 below shows the IL-1 production inhibitory activity of the compound of the present invention (Compound of Production Example 2).

[0048]

[Table 3]

As control compounds 1 and 2, tenidap (5-chloro-3- (2-thenoyl) -2-oxyindole-1-carboxamide) and prenidosolone were used, respectively. The -1 production inhibitory activity effect was superior to that of tenidap. In addition, it was confirmed that the 2-ethynylmalonic acid derivative according to the present invention (the compound of Production Example 2) exhibits dose-dependent production inhibition at a concentration of 100 μg / ml or less.

(2) Human Peripheral Blood Monocyte Cytotoxicity Measurement After collecting a part of the supernatant in (1), human monocyte cells were stained with trypan blue, and the whole cells were examined under a microscope using a cell counting plate. The viable cells (%) in the cells were measured. According to it, the 2-ethynylmalonic acid derivative according to the present invention (the compound of Production Example 2) showed no significant toxicity to cells at a concentration of 100 μ / ml or less which suppressed IL-1 production.

(3) Effect on IL-1β production from human leukocytes (Preparation of human leukocyte suspension) Human peripheral blood was centrifuged at room temperature (900 rpm, 15 minutes) to remove the platelet fraction in the supernatant. Then, the buffy coat formed on the red blood cell fraction was collected. Three times the amount of Hanks 'solution (Nissui) was added to the suspension, suspended on Ficoll solution, cold Hanks' solution was added and resuspended, and then centrifuged at 4 ° C (2000 rpm, 10 minutes), and the supernatant was obtained. Was removed. After this operation was repeated 3 times, an appropriate amount of Hanks' solution was added and resuspended, and this was used as a leukocyte suspension in the experiment. (Measurement method) Leukocyte suspension prepared from human peripheral blood and DM
The inhibitors dissolved in SO were mixed in Hank's solution and then preincubated at 37 ° C. for 30 minutes. Then, add LPS (100 μg / ml) and mix at 37 ° C for 3
Reacted for hours. After stopping the reaction by ice-cooling, the mixture was centrifuged (2000 rpm, 10 minutes), and the IL-1β concentration in the supernatant was determined by the human IL-1β ELISA kit (Quantikine; R & D System).
s). Calculate the inhibition rate by the following formula,

[0052]

[Equation 1]

The results are shown in Table 4.

[0054]

[Table 4]

Compound 3 having a high inhibition rate among these compounds
The activity of the dots at lower concentrations was measured and is shown in Table 5.

[0056]

[Table 5]

[0057]

According to the above test results, the 2-alkynylmalonic acid derivative according to the present invention has an activity close to that of prednisolone which is a conventional excellent steroid compound.
In addition, it is clear that it has an inhibitory activity equivalent to or higher than that of tenidap, which is a typical example of non-steroidal compounds, so when it is used as a medicine, it has been used for rheumatoid arthritis and inflammatory diseases. The non-steroidal anti-inflammatory drug (NSAID) is not a symptomatic treatment, but is expected to have a suppressive effect on the progression of the disease state. In addition, the 2-alkynyl malonic acid derivative according to the present invention is
Osteoarthritis, gout, atherosclerosis, atopic dermatitis, lung disease with granulomas, various encephalitis, diabetes, osteoporosis, and cells that produce IL-1 and express HLA-DR antigen That is, the effect can be expected as a cell function inhibitor selective for antigen presenting cells, that is, an immunosuppressant.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 31/695 C07C 69/734 Z 9279-4H 229/12 7537-4H 229/30 7537-4H 233 / 47 7106-4H 233/54 7106-4H 271/22 9451-4H C07D 309/12 C07F 7/10 A

Claims (4)

[Claims] 1. The following general formula [I]: (In the formula, R represents a hydrogen atom, an optionally substituted aryl group or aralkyl group, a cycloalkyl group, a cycloalkenyl group, an optionally substituted lower alkyl group or a tri-lower alkylsilyl group, and R ¹ is A hydrogen atom or a carboxyl protecting group, and R ² represents a hydroxyl group, a lower alkoxy group, an optionally protected amino group or an optionally protected alkylamino group) or a 2-alkynylmalonic acid derivative represented by An interleukin-1 production inhibitor comprising a salt. 2. An interleukin-1β comprising the 2-alkynylmalonic acid derivative according to claim 1 or a salt thereof.
Production inhibitor. 3. A therapeutic agent for rheumatoid arthritis comprising the 2-alkynylmalonic acid derivative according to claim 1 or a salt thereof. 4. A therapeutic agent for autoimmune diseases, which comprises the 2-alkynylmalonic acid derivative according to claim 1 or a salt thereof.